Method of making a personalized bone graft

ABSTRACT

An anatomically-shaped, human bone graft may be cultivated ex vivo using a bioreactor capable of perfusing large complex porous scaffolds. Scaffolds derived from image-based modeling of a target are seeded with human mesenchymal stem cells and cultivated. A bioreactor configured to house complex three-dimensional scaffold geometries provides controlled flow for perfusion of the cells. Dense uniform cellular growth can be attained throughout the entire scaffold as a result of the medium perfusion. In an embodiment, the bioreactor has a mold into which perfusion medium is pumped under pressure and multiple ports through which the medium exits the mold.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/633,317 filed Jun. 26, 2017, which is a divisional application ofU.S. patent application Ser. No. 13/148,735 filed Oct. 28, 2011 which isa National Stage Entry of International Patent Application No.PCT/US10/26120 filed Mar. 3, 2010, which claims the benefit of U.S.Provisional Application No. 61/157,019, filed Mar. 3, 2009, U.S.Provisional Application No. 61/249,999, filed Oct. 9, 2009, and U.S.Provisional Application No. 61/250,166, filed Oct. 9, 2009, all of whichare hereby incorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. Government support under Grant No. R01DE161525-01 and P41 EB02520-01A1 awarded by the National Institutes ofHealth (NIH). The U.S. Government has certain rights in this invention.

FIELD

The present disclosure relates generally to tissue engineering and, moreparticularly, to methods, devices, and systems for bone tissueengineering.

BACKGROUND

Bone reconstructions often involve autologous tissue grafting. In such aprocedure, bone from one part of a patient is used to replace missing ordamaged bone in another part of the patient. Because the bone graft istaken from the patient's own body, there is a reduced risk of thepatient's body rejecting the graft. However, autologous tissue may belimited due to harvesting difficulties, donor site morbidity, and/or aclinician's ability to contour delicate three dimensional shapes.

SUMMARY

The availability of personalized bone grafts engineered from thepatient's own stem cells may revolutionize the way bone defects arecurrently treated. A ‘biomimetic’ approach utilizes stem cells,regulatory factors, and appropriate scaffolds to guide celldifferentiation and assembly into the desirable tissue phenotypes. Thus,an anatomically-shaped, human bone graft may be cultivated ex vivo usinga bioreactor capable of perfusing large complex porous scaffolds.Scaffolds derived from image-based modeling of a target can be seededwith human mesenchymal stem cells (hMSCs) and cultivated. The bioreactorencloses the scaffold and controls flow for perfusion of the cells.Dense uniform cellular growth can be attained throughout the entireconstruct as a result of the medium perfusion. In embodiments, thebioreactor has a mold into which perfusion medium is pumped underpressure and ports at multiple sites through which the medium can enterand/or exit the mold.

In embodiments, a method of making a bone graft can include shaping ascaffold according to a target shape of bone to be replaced, forming asupport with a cavity which closely conforms to the scaffold resultingfrom the shaping, and pumping a perfusate into the cavity whilesimultaneously receiving perfused perfusate through outlets sealed atmultiple points to and about the scaffold. The multiple points can beseparated and arranged such that the perfusate enters the scaffold overa substantial surface thereof and exits the scaffold at the multiplepoints.

In embodiments, a bone graft can include a scaffold having cells. Thecells can be arranged such that they have a density pattern that isresponsive to a flow pattern of perfusate through the scaffold. The flowpattern can include a gradient of decreasing cell density stemming fromfocuses at a surface of the scaffold.

In embodiments, a method of making a tissue structure can includeforming an image of a target tissue structure, shaping athree-dimensional scaffold responsively to the image, and seeding thescaffold with cells. The method can also include delivering nutrients tothe cells within and on the surface of the scaffold by flowing anutrient fluid into a tightly conforming vessel holding the scaffold,through multiple first surface portions of the scaffold, and out throughat least one second surface portion.

In embodiments, a tissue engineering system can include a machiningdevice, a bioreactor, and a flow mechanism. The machining device can beconfigured to machine a three-dimensional vessel with an internalsurface closely following a shape of a target anatomy of a patient. Thebioreactor can have a recess configured to receive said vessel andoutlet ports configured to accept at least one lumen and to permit flowcommunication between the at least one lumen and an internal volumedefined by the internal surface of said vessel. The flow mechanism canbe configured to remove a perfusate from the at least one lumen andreturn it to the internal volume of the vessel.

In embodiments, a method for making a bone tissue structure can includeseeding a porous scaffold with mesenchymal stem cells, and perfusingculture medium throughout an interstitial volume of the porous scaffoldfor a period of time such that the mesenchymal stem cells developlamellae of bone tissue which fills pore spaces of the scaffold.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B are an images of a human jawbone showing a location of a TMJcondyle with respect thereto.

FIG. 1C is an image of a de-cellularized scaffold, according to one ormore embodiments of the disclosed subject matter.

FIG. 1D is an image of an assembled bioreactor, according to one or moreembodiments of the disclosed subject matter.

FIG. 1E is a schematic diagram showing a bioreactor with a scaffoldcontained therein, according to one or more embodiments of the disclosedsubject matter.

FIG. 1F is a side view of a three-dimensional model of a bioreactor witha scaffold contained therein, according to one or more embodiments ofthe disclosed subject matter.

FIGS. 1G-1H and 1J show various steps in the assembly of a bioreactor,according to one or more embodiments of the disclosed subject matter.

FIG. 1K is a schematic diagram of an apparatus for perfusion support fora bioreactor, according to one or more embodiments of the disclosedsubject matter.

FIG. 2 is a chart showing cultivated cell count under variousconditions, according to one or more embodiments of the disclosedsubject matter.

FIGS. 3A through 3H are images of exemplary bone constructs formed undervarious conditions, according to one or more embodiments of thedisclosed subject matter.

FIGS. 4A through 4C are images of a bone scaffold illustrating changesin the scaffold due to perfusion over time, according to one or moreembodiments of the disclosed subject matter.

FIGS. 5A-5B show computational models of perfused flow in a scaffold inthe bioreactor, according to one or more embodiments of the disclosedsubject matter.

FIGS. 5C through 5F are images showing characteristics of cultivatedtissue grafts, according to one or more embodiments of the disclosedsubject matter.

FIG. 6A is an image showing a von Kossa stain of histological sectionsof pellets cultured under osteogenic conditions, according to one ormore embodiments of the disclosed subject matter.

FIG. 6B is a chart showing calcium content normalized to DNA values ofcell pellets cultured for four weeks in osteogenic (ost), chondrogenic(ch), or control (ctr) media.

FIG. 6C is an image showing alcian blue stains of cell pellets culturedunder chondrogenic conditions.

FIG. 6D is an image showing oil Red 0 stains of lipid droplets inpellets cultured under adipogenic conditions.

FIG. 7A is an image of a spinner-flask apparatus used to seed cells intoscaffolds, according to one or more embodiments of the disclosed subjectmatter.

FIG. 7B is an image showing H & E stains of scaffolds one day afterseeding. FIG. 7C is an image showing live-dead stains of scaffolds oneday after seeding.

FIGS. 8A through 8D are images showing the effects of perfusion flowrate on cell density, according to one or more embodiments of thedisclosed subject matter.

DETAILED DESCRIPTION

The availability of personalized bone grafts engineered from a patient'sown stern cells has the potential to alter the way bone defects arecurrently treated. Bone grafts with a high degree of shape fidelity canbe produced, which have a low risk of rejection by the patient's body.The functionality of engineered bone grafts can be evaluated by themechanical properties and the ability of cells to make tissue specificproteins. Craniofacial bone grafts also have the characteristic thattheir functionality is linked to their overall geometry.

Bone grafts of high utility for reconstructive surgery can be based on“designer scaffolds” shaped into gross geometries specific to thepatient and the defect being treated. Anatomically shaped, viable humanbone grafts can be engineered using human mesenchymal stem cells (hMSCs)and a “biomimetic” scaffold bioreactor system. The disclosed techniquesmay be used to engineer tissue structures such as bone grafts, includingbut not limited to autografts. The hMSCs may be suited for use incranial and maxillofacial

applications due to their easy accessibility, capability for in vitroproliferation, and the potential to form cartilage, bone, adipose andvascular tissues.

The potential of hMSCs for differentiation potential along mesenchymallineages may be characterized for each batch of cells by culturing cellpellets under osteogenic, chondrogenic, and adipogenic conditions for aculture period of time, for example, four weeks. FIG. 6A shows a vonKossa stain of histological section of hMSC pellets cultured underosteogenic conditions. FIG. 6B is a graph illustrating the calciumcontent of hMSC pellets cultured for four weeks in osteogenic (ost),chondrogenic (ch), or control (ctr) medium. The data in FIG. 6B has beennormalized with respect to DNA values. FIG. 6C represents an alcian bluestain of hMSC pellets cultured under chondrogenic conditions, while FIG.6D is an image of an oil red O stain of liquid droplets of hMSC cellpellets cultured under adipogenic conditions. Thus, hMSCs have beenpre-differentiated along chondrogenic and osteogenic lineages. Moreover,the hMSCs can form distinct osseous and cartilaginous regions. However,other types of stem cells may also be used according to one or morecontemplated embodiments.

In vitro control of cell viability and tissue development in clinicallysized and shaped bone tissue constructs determines their utility forregenerative medicine. The enhancement of mass transport and thegeneration of hydrodynamic shear, which are important for bonedevelopment and function, may require interstitial flow. Thus, describedherein are tissue engineering devices, systems, and methods for creatingin vitro an entire bone condyle containing viable cells at physiologicdensity and well-developed bone matrix. In embodiments, hMSCs can beinduced to form bone on a de-cellularized scaffold that has the exactgeometry of the desired bone structure. For example, the desired bonestructure may be a temporomandibular joint (TMJ) condylar bone, ascircled in FIGS. 1A-1B.

The hMSCs can be induced to form bone on the scaffold using an“anatomical” bioreactor with control of interstitial flow. Flow patternsassociated with the complex geometry of the bone graft provide a uniqueopportunity to correlate the architecture of the forming bone withinterstitial flow characteristics, under controllable in vitroconditions. This approach can help provide a variety of anatomicallyshaped bone grafts designed to meet the needs of a specific patient anda specific craniofacial or orthopedic reconstruction. Other applicationswill also be evident from the present disclosure.

In embodiments, anatomically-shaped scaffolds may be generated by CNCmachining fully de-cellularized (e.g., trabecular) bone based ondigitized images of the desired bone structure. For example, thetrabecular bone may be derived from the subchondral region of the kneejoint of a calf and subsequently treated to remove any cellularmaterial. The bone may be washed with high velocity water to removemarrow and then subject to a wash for 1 hour in PBS with 0.1% EDTA (w/v)at room temperature. The bone may then be subject to sequential washesin hypotonic buffer (10 mM Tris, 0.1% EDTA (w/v)) overnight at 4° C.,detergent (10 mM Tris, 0.5% SDS (w/v)) for 24 hours at room temperature,and enzymatic solution (50 U/mL DNAse, 1 U/mL RNAse, 10 mM Tris) for 3-6hours at 37° C. to remove any remaining cellular material.Alternatively, other types of scaffold material may be used. Forexample, the scaffold may be formed from other naturally occurringmaterial such as coral, a synthetic material such as a ceramic orpolymer, and/or other natural or synthetic porous structures.

In embodiments, the de-cellularized trabecular bone can be seeded withhMSCs and cultured with interstitial flow of culture medium. Abioreactor with an internal chamber in the exact shape of the desiredhuman bone (e.g., a human TMJ) controls perfusion throughout theengineered scaffold. After a cultivation period (e.g., 5 weeks), tissuegrowth can be evidenced by the formation of confluent layers of lamellarbone (by scanning electron microscopy), markedly increased volume ofmineralized matrix (by quantitative microcomputer tomography), and theformation of osteoids (histologically). Experiments have shown thatcells in such a construct are fully viable at a physiologic density,which is a desirable property in grafts. Moreover, the density andarchitecture of bone matrix correlated with the intensity and pattern ofthe interstitial flow, as determined in experimental and modelingstudies.

In an embodiment, the anatomical shape of a bone structure, for examplethe TMJ of a patient, can be defined from digitized medical images, suchas the circled portions in FIGS. 1A-1B. The TMJ has clinical importanceand a complex shape, which may be desirable for evaluating anddemonstrating the tissue engineering techniques and devices describedherein. However, the techniques and devices described herein are equallyapplicable to other types of bones and shapes as well. The shape of thebone structure can be faithfully reproduced by machining ade-cellularized trabecular bone scaffold. For example, the scaffold canbe prepared by milling the de-cellularized bone based on clinicalcomputerized tomography (CT) images of the patient representing theexact geometry of the human TMJ condyle. An example of such a milledscaffold for a TMJ condyle is shown in FIG. 1C. The CT image data can befed to an appropriate computer-assisted machining device in order toform an appropriately shaped scaffold from fully de-cellularizedtrabecular bone. Although the shape of a TMJ condyle has been shown anddiscussed for the scaffold, other shapes are of course possibledepending on the desired bone graft.

The hMSCs may be cultured up to the 3r^(d) passage and then used forseeding the scaffolds. After, seeding, the scaffolds may be culturedwith osteogenic medium, such as Dulbecco's Minimal Essential Medium(DMEM) supplemented with 10% fetal bovine serum (FBS), 1%penicillin-streptomycin, 100 nM dexamethasone, 10 mMbeta-glycerophosphate, and 50 mM ascorbic acid-2-phosphate. The machinedscaffold can be seeded in a stirred suspension of hMSCs, for example, ata density of 10⁶ cells/ml. The scaffold may then be preculturedstatically for an additional period of time, for example, 1 week, toallow for cell attachment. The cell-seeded scaffolds may then betransferred into the “anatomical” bioreactor chambers and hydrodynamicshear applied by starting the medium perfusion. Perfusion using thebioreactor can then be performed for an additional period of time, forexample, 4 weeks.

FIGS. 1D-1F illustrate a perfusion bioreactor system according to anembodiment of the present disclosure. The perfusion bioreactor system isdesigned to control the perfusion path through a geometrically complexscaffold 100. An anatomically shaped mold 102 holds the scaffold 100.Because of its modular design, the bioreactor chambers can accommodatedifferent geometries by simply inserting a different mold 102 ofappropriate shape, created, for example, by using digitized medicalimages. In addition, the bioreactor can be made from materials whichallow for noninvasive visualization and monitoring of mediumdistribution within the tissue scaffolds throughout the cultivationperiod

The interior of the bioreactor is designed to conform to the surface ofthe scaffold. For example, the interior of the bioreactor can be formedusing a polydimethylsiloxane (PDMS) mold. The PDMS mold can be createdby pouring PDMS around a CNC-milled piece of delrin (acetal copolymer)generated from the digital images so as to exactly duplicate the shapeof the bone scaffold 100. Once the PDMS has cured, the delrin isremoved. The scaffold 100 can then be placed into the PDMS mold 102 andinserted into the bioreactor. The PDMS mold 102 thus forms an innercavity within inner chamber 108 for holding the scaffold 100 therein.Although molding of PDMS has been described, other materials andtechniques may also be employed to produce the closely-conforming innercavity housing the scaffold within the bioreactor.

A system providing software for generating machine instructions forfabricating the scaffold and/or the holding fixture (in examples,corresponding to mold 102) may also be provided. The software may takeimages of the target anatomy and produce instructions for machining theholding fixture or vessel to have close conforming walls as describedwith respect to mold 102 above. The system could also be provided with amilling device for making a positive structure to create the mold or anegative structure for the mold itself.

Other types of “machining” are also possible, such as, but not limitedto, 3-D printing or rapid prototyping/fabrication systems (e.g.,computer guided photopolymerizing device).

FIGS. 1G-1H and 1J show steps in the assembly of the exemplarybioreactor. A bone scaffold 100 is assembled with the mold 102 andplaced into the inner chamber casing 108 of the bioreactor. The innerchamber casing 108 may be made of, for example, polypropylene orpolystyrene. A metal rod 106 is placed through preformed holes in themold 102 and inner chamber casing 108 so as to align the assembly withgroove 110 in outer chamber 112. Outer chamber 112 may be made of, forexample, clear acrylic. For example, the outer chamber of the bioreactorcan have an external diameter of 7.5 cm and a height of 5 cm. However,other suitable materials and sizes may also be used for the bioreactoraccording to one or more contemplated embodiments. By virtue of thealignment provided by rod 106, the assembly and the scaffold therein canbe maintained in the correct orientation, as shown in FIG. 1H. Theassembly of the scaffold 100, mold 102, and inner chamber casing 108 isinserted into the outer chamber 112, which is then tightly capped with atop 114. The top 114 may be made of, for example, polyetherimide (PEI).Top 114 may also have a groove therein so as to accommodate rod 106 whensealing the inner chamber 108 and outer chamber 112.

The outer chamber 112 and cap 114 also serve to compress mold 102 aroundthe scaffold 100, thereby forcing culture medium to flow through theentire scaffold rather than channeling around the periphery thereof. Theouter chamber 112 can have a plurality of radial cylindrical ports, forexample, holes 104. For example, the outer chamber 112 can have sixports arranged equidistant around the circumference of the outer chamber(i.e., at 60° intervals). Each of the cylindrical ports can serve as aguide for controlling the exact position and depth for the insertion ofa needle 116 into scaffold 100, the purpose of which is discussed ingreater detail below. For example, needle 116 may be a 23 gauge needle.Snugly fitting delrin rings may be placed into each hole 104. The centerof the delrin rings may be tapped so as to accommodate nylon screwswhich have been cored to fit the 23 gauge needles therein. The needlesmay be place into the nylon screws such that the ends of the needlesprotrude from the screws. When screwed into the delrin rings, the nylonscrew assembly allows the needle to penetrate into the scaffold in theinner chamber to serve as an inlet/outlet port for medium flow.

In an alternative, one or more outlet ports of may be provided in andaround the scaffold to evenly distribute fluid through the scaffold andallow full perfusion of the interstitial areas of the scaffold. Suchoutlet ports may be smaller than the inlet port. In still anotheralternative, one or more of the needle ports 116 can serve as an inletwhile connector 118 simultaneously serves as an outlet for perfusing thescaffold. In yet another alternative, one or more of the needle ports116 can serve as an inlet while connector 118 simultaneously serves asan inlet for perfusing the scaffold.

The location of each hole 104 may be determined, for example, usingcomputer-aided design based on a three-dimensional reconstruction of thedesired bone structure. In embodiments, three of the six ports 104 canbe used as outlets. An additional port, aligned with the central axis ofthe inner chamber 108, can be connected to tubing 122 via a connector118 (e.g., a luer connector). This central port can serve as a singleinlet for medium to enter scaffold 100. Thus, flow can enter via tubing122 into the inner chamber 108, perfuse through scaffold 100, and exitthrough three (or more) needle outlets 116. Tubing 120 connected to theneedle outlets 116 can convey the perfused fluid therefrom. In analternative, the flow of medium may be reversed such that culture mediumenters the mold 102 through needles 116, perfuses through scaffold 100,and exits via tubing 122.

The flow rate of medium exiting through outlet ports 104 via tubing 120can be regulated such that the flow rate for each outlet is equal. Suchregulation may be accomplished, for example, by adjusting clamps ontubing 120. Alternatively, inline flow regulators or valves may be usedto control the flow rate of each outlet. Of course, other flowregulation mechanisms are also possible according to one or morecontemplated embodiments. Moreover, the flow rate for each outlet neednot be equal. Rather, the flow rate may be controlled to achieve adesired flow profile conducive to cell growth as determined fromcomputer-aided flow modeling and/or experimentation.

As shown in FIG. 1K, culture medium perfused through the bioreactor 1004can be conveyed via pump 1010 back into a reservoir 1002 for reuse. Thereservoir 1002 may also serve as a bubble trap with an air outlet 1008.The pump 1010, which may be, for example, a low-flow multi-channeldigital peristaltic pump, can recirculate the culture medium from thereservoir 1002 into the bioreactor. The medium in the reservoir 1002 maybe changed periodically through port 1006 of the reservoir. For example,a syringe can be attached to port 1006 to sterilely remove culturemedium from and add culture medium to the reservoir without disturbingthe operation of the bioreactor 1004. The reservoir may contain aninitial volume of, for example, 40 ml of culture medium. Half of theculture medium in the reservoir may be replaced, for example, everythree days.

Tissue engineering of large bone constructs requires flow through theinterstices and/or pores of the scaffold for efficient transport ofnutrients and waste materials between the cells and culture medium. Inaddition, interstitial flow allows for direct exposure of cells tohydrodynamic shear, which may be important for osteogenesis. Thevolumetric flow-rate (e.g., 1.8 ml/min) and the correspondingsuperficial velocity (e.g., an average of 0.06 cm/s) can be selected tosustain dense tissue growth throughout the scaffold. For example, theflow-rates may be selected so as to be within the range of flow-ratesand superficial velocities that stimulate osteogenic differentiation ofhMSCs.

The cells can be cultured in a scaffold that has the structural,biochemical and mechanical properties of native bone and the actualgeometry of the final graft. For example, the scaffold can be formedfrom fully de-cellularized bone, which has been machined, for example,by image-guided fabrication, to achieve the desired geometry of thefinal graft. The void volume of such a de-cellularized bone wasdetermined by micro-CT analysis to be greater than 80%. SEM andhistological analysis of de-cellularized bone also revealed pore sizesof approximately 1 mm. Such structural features may enable efficient andspatially uniform dynamic seeding of hMSCs into the scaffolds.Histological evaluation of freshly seeded scaffolds demonstrated thathMSCs lined the internal pore walls, while leaving pore spacesunobstructed.

Referring now to FIG. 7A, a spinner flask (cap shown with TMJconstructs) can be used to seed cells into TMJ scaffolds. FIG. 7B show ahemotoxylin and eosin (H&E) stain of a scaffold one day after seeding.As is evident from FIG. 7B, pore spaces in the scaffold remain open, andcells can be found only along the walls of the pores (see arrows in FIG.7B). FIG. 7C shows a live-dead stain of a scaffold one day afterseeding. As is evident from FIG. 7C, a high percentage of viable cellscan be retained throughout the seeding process.

After one hour, the seeding efficiency was found to be 34.0±7.1%,resulting in approximately 3.4×10⁶ cells per construct attaching in aspatially uniform manner. Scaffolds were cultured statically for oneweek prior to placing in the bioreactors, enabling firm cell attachmentand deposition of extracellular matrix before the exposure tohydrodynamic shear forces. Of course, other methods and devices forseeding of the scaffolds are contemplated.

After seeding, the scaffold may be assembled under sterile conditionsinto the bioreactor, as discussed with respect to FIGS. 1G-1H and 1J.Quick assembly can maintain cell viability in the scaffold throughoutthe process. Using the bioreactor, culture medium can be perfusedthroughout the entire scaffold. For a given flow rate, equal outlet flowrates are maintained at each of the three outlets, although differentialoutlet flow rates are also possible. The optimal inlet flow rate couldbe determined based on fluid flow analysis, computer-aided modeling, orexperimental data. For example, the inlet flow rate may be between 0.4ml/min and 1.8 ml/min. Experimental evaluation of cell density anddistribution after 5 weeks of culture suggests that 1.8 ml/min may yieldthe best cell distribution and most rapid tissue development for thedisclosed bioreactor. Based on micro-CT analysis, the averagecross-sectional area through the scaffolds in the direction of flow wasdetermined to be 0.5 cm².

For this cross-sectional area, the 1.8 ml/min inlet flow ratecorresponds to an average superficial flow velocity of 0.06 cm/s.However, other flow rates and/or superficial flow velocities may bechosen depending on, for example, bioreactor geometry, stem cell type,scaffold size, scaffold type, and/or pore size.

Due to the complex distribution of flow within the tissue scaffolds,flow rates as high as 0.15 cm/s are possible in certain scaffoldregions, and as low as 0.0001 cm/s in other scaffold regions. In thewhole range of these flow velocities, hMSCs may maintain completeviability and exhibit characteristics of osteogenic differentiation.There is also no apparent threshold in fluid flow rate after whichperfusion becomes detrimental to hMSCs. It is therefore possible thattissue growth can be further improved by increasing the flow rates inthe bioreactor above the 1.8 ml/min inlet flow rate described above.

Final cell densities were approximately 105-210×10⁶ cells/ml. Such highcell densities may be important for functional bone tissue formation forcell-cell interaction. For statically cultured constructs, the loosepacking of cells (indicated by SEM) and only minimal osteoid formation(indicated by histology) provided evidence of limited functionaldifferentiation of the hMSCs in the inner regions of these constructs.For bioreactor cultured constructs, various imaging modalities,discussed in detail below, confirmed that cells formed dense tissuesthroughout the construct volumes, leading to larger increases in bonevolume.

Referring to FIGS. 8A through 8D, the effects of perfusion flow rate oncell density in a scaffold are shown. FIGS. 8A-8D are H&E stains of thecentral regions of scaffolds cultivated for 5 weeks under various flowconditions. FIG. 8A was cultivated under a static (i.e., no flow)condition. FIG. 8B was cultivated under a 0.4 ml/min inlet flow rate.FIG. 8C was cultivated under a 0.8 ml/min inlet flow rate. FIG. 8D wascultivated under a 1.8 ml/min inlet flow rate. As is evident from FIGS.8A-8D, increasing the flow rate led to increasingly dense matrixdeposition in the pore spaces.

By perfusing the scaffold in the disclosed manner using the bioreactor,cells are able to proliferate in the scaffold so as to form a viablebone graft. In experiments, cells proliferated extensively over thefirst week of culture, as evidenced by an approximately 7.5-foldincrease in DNA content. The DNA content continued to increasethroughout the cultivation period under both static (4.5 fold increase)and perfused (10 fold increase) culture conditions resulting in overall37 and 75 fold increases, respectively, in cell numbers relative toinitial seeding values. Referring now to FIG. 2, cell numbers increasedwith time of culture and medium perfusion. From day 1 to day 7, the cellnumbers increased 7.5-fold, from 3.4×10⁶ to 25×10⁶ cells/scaffold. Overthe subsequent 4 weeks, cell numbers in static culture increased4.5-fold, to approximately 110×10⁶ cells/scaffold, whereas the increasein perfused bioreactor culture was 10-fold, to approximately 250×10⁶cells/scaffold.

Referring now to FIGS. 3A through 3H, bone formation was also markedlyenhanced by perfusion in a manner responsive to the fluid flow pattern.FIGS. 3A-3D show scaffolds cultured under static conditions while FIGS.3E-3H show scaffolds cultured with medium perfusion in the disclosedbioreactor. FIGS. 3A and 3E show trichrome staining of entirecross-sections of scaffolds, which illustrate differences in the newmatrix distribution compared to the original scaffold for the static(FIG. 3A) and perfused (FIG. 3E) culture groups. Moreover, significantdifferences can be observed in osteoid formation (arrows) in the centralregions of scaffolds cultured statically (FIG. 3B) and in perfusion(FIG. 3F).

FIGS. 3C, 3D, 3G, and 3H are SEM images of the central scaffold regions.In particular, FIGS. 3C and 3D show that statically cultured scaffoldsexhibit empty pore spaces and loosely packed cells. In contrast, FIGS.3G and 3H show that scaffolds cultured in perfusion demonstrateformation of dense and confluent lamellae of bone tissue that filled theentire pore spaces. Measured increases in cell numbers with time andculture regimen have been corroborated with imaging data. Scaffoldscultivated under static conditions formed new matrix primarily at theperiphery (FIG. 3A), whereas bioreactor-grown scaffolds displayed newtissue growth throughout their volumes (FIG. 3E). Histological sectionsdemonstrated stark contrast in cell grmvth and osteoid formationpatterns in the central regions between the two culture groups (FIGS. 3Band 3F). The new osteoid area normalized to existing bone trabeculae inthe central regions of static scaffolds was 0.031±0.013 mm²/mm² ascompared to 0.210±0.022 mm²/mm² for perfused scaffolds (i.e., a 7-foldincrease due to perfusion). SEM images of the inner regions of thetissue scaffolds corroborated these findings yet were uniquelyinstructive. The inner regions of static scaffolds showed pore spacesthat were empty or only loosely packed with the cells and matrix (FIGS.3C and 3D), in contrast to perfused scaffolds, which showed denselypacked pore spaces throughout their entire volumes (FIGS. 3G and 3H).

FIGS. 4A through 4C are reconstructed images of a bone scaffold taken bya three-dimensional micro-CT illustrating changes in the scaffold forvarious perfusion times. FIG. 4A was taken at a time at the beginning ofthe perfusion process, FIG. 4B in the middle of the perfusion process,and FIG. 4C near the end of the perfusion process. In particular, thesefigures illustrate the development of the architecture of themineralized bone matrix of the scaffold over time and in a mannerdependent on culture conditions. The images thus demonstrate the changesin pore structure (relative to the initial state) that were evident atthe end of the 5-week cultivation period. Bioreactor scaffolds exhibitedmore rapid deposition of new mineral matrix as compared to staticscaffolds. The mineral deposition in pore spaces is also evident fromFIGS. 4A-4C. Statistically significant increases in bone volume wereobserved with time of culture in both static (8.7%) and perfused (11.1%)scaffolds, with consequent increases in trabeculae number (Tb. N*) andthickness (Tb. Th), and decreases in trabecular spacing (Tb. Sp*). Thestructural model index (SMI) numbers for static and perfused scaffoldswere lower after cultivation indicating a trend toward the formation ofplate-like trabeculae.

Computer-aided modeling can be performed to evaluate the effect ofperfusion flow parameters. An example of such a computer model for a TMJscaffold in the disclosed bioreactor is shown in FIGS. 5A-5B. Inparticular, theoretical modeling of the flow in the scaffold indicated awide distribution in the magnitude (0 to 0.15 cm/s) and directions offlow velocities within the constructs. The flow rates were highest inthe inlet and outlet regions, adjacent to the needle ports. Due to thecomplex geometry of the scaffold, its flat base is not at the center ofthe chamber, resulting in spatial gradients of flow distribution acrossthe base. The lowest flow rates occurred at far-right and far-leftprojections of the scaffolds with the velocity vector computationindicating near zero flow at the extremities (FIG. 5A), since the outletneedle was not placed at the tip of the projection. Dye studies showedthat medium does in fact perfuse these extreme regions. Histologicalanalysis of 5-week scaffolds clearly demonstrated cell survival andmatrix production in these regions.

Referring now to FIGS. 5A-5F, bone matrix morphology has been correlatedto the patterns of medium perfusion flow. FIG. 5A shows color-codedvelocity vectors indicating the magnitude and direction of flow throughthe entire scaffold based on experimentally measured parameters. FIG. 5Bis digitally sectioned, and the color-coded contours are used toindicate the magnitude of flow in the inner regions. Scanning electronmicroscopy (SEM) images demonstrated morphological variations in thetissue morphology with the variation in fluid flow pattern. For example,in the middle regions where the fluid flow is unidirectional, tissueappears smooth and aligned, and the crystalline structures can be easilyseen on the surface (FIGS. 5C-5D). At the base of the projection, closeto the outlet port, the model indicates large local changes in thevelocity vector, effectively resulting in swirling flow patterns. Highmagnification SEM images of this region demonstrated a corresponding“swirling” of mineralized matrix structure (FIGS. 5E, F).

Thus, the approach disclosed herein demonstrates that it is possible tocreate bone grafts using a bioreactor that (i) houses anatomicallyshaped tissue scaffolds with complex geometries, (ii) providescontrolled interstitial flow of culture media through the pore spaces ofthe scaffolds, and (iii) enables the establishment of cultivationprotocols for engineering large human bone grafts.

In embodiments, several tissue engineering operations may be employedincluding, but not limited to: (i) imaging guided fabrication ofanatomical scaffolds; (ii) use of de-cellularized bone as anosteo-inductive scaffold; (iii) use of multi-potent mesenchymal stemcell populations, applicable in either autologous or allogeneic fashion;(iv) perfusion based environmental control and biophysical stimulationof cultured bone constructs; and (v) a computational modelingoptimization of bioreactor design.

In embodiments, a method for bone tissue engineering can include,separately or in combination: (i) imaging at least a portion of apatient for a desired bone graft; (ii) machining a porous scaffold intothe shape of the desired bone graft; (iii) seeding the porous scaffoldwith hMSCs; and (iv) perfusing culture medium throughout an interstitialvolume of the porous scaffold for a period of time such that the hMSCsdevelop lamellae of bone tissue which fill the pore spaces of thescaffold.

The methods, systems, and devices for tissue engineering describedherein thus enable the formation of geometrically complex boneconstructs of high structural and biological fidelity. Computationalmodeling of fluid flow may also provide important insights into tissueresponses to biophysical stimuli. Although particular configurationshave been discussed herein, other configurations can also be employed.Furthermore, the foregoing descriptions apply, in some cases, toexamples generated in a laboratory, but these examples can be extendedto production techniques. For example, where quantities and techniquesapply to the laboratory examples, they should not be understood aslimiting. In addition, although the production of bone tissue graftshave been specifically described herein, the techniques described hereinare applicable to other tissues as well.

Features of the disclosed embodiments may be combined, rearranged,omitted, etc., within the scope of the invention to produce additionalembodiments. Furthermore, certain features may sometimes be used toadvantage without a corresponding use of other features.

It is, thus, apparent that there is provided, in accordance with thepresent disclosure, methods, devices, and systems for bone tissueengineering using a bioreactor. Many alternatives, modifications, andvariations are enabled by the present disclosure. While specificembodiments have been shown and described in detail to illustrate theapplication of the principles of the invention, it will be understoodthat the invention may be embodied otherwise without departing from suchprinciples. Accordingly, Applicants intend to embrace all suchalternatives, modifications, equivalents, and variations that are withinthe spirit and scope of the present invention.

What is claimed is:
 1. A tissue engineering system comprising: amachining device configured to machine a three-dimensional vessel withan internal surface closely following a shape of a target anatomy of apatient; a bioreactor having a recess configured to receive said vesseland outlet ports configured to accept at least one lumen and permit flowcommunication between the at least one lumen and an internal volumedefined by the internal surface of said vessel; and a flow mechanismconfigured to remove a perfusate from the at least one lumen and returnit to the internal volume of the vessel.
 2. The system of claim 1,wherein the machining device is configured to shape the vessel internalsurface responsively to a target anatomy of a patient.
 3. The system ofclaim 1, wherein the flow mechanism includes a pump.